This project, targeted for the National Human Genome Research Institute, will apply nearly a dozen new technologies to improve the performance of DNA chip microarrays as they detect, quantitate, and characterize nucleic acids. The technologies include: An artificially expanded genetic information system (AEGIS), a DNA-like molecular recognition system that forms duplexes following Watson-Crick rules, but does not interact with natural nucleic acids. A self-avoiding molecular recognition system (SAMRS), whose components do bind to natural DNA but do not bind to other components of the same unnatural SAMRS system. DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS Reversible terminators, which terminates primer extension but (unlike dideoxynucleoside triphosphate terminators) can be later removed to continue primer extension, and polymerases that accept them. These support "sequencing using cyclic reversible termination" to validate array hits while they are still on the-array. SNAP2 technology that primes DNA and RNA with the specificity of a 16mer but the discriminatory power of 8mers [Lea06]. Novel technology for on-array DNA synthesis using an inkjet DNA array synthesizer. Convertible nucleotides that enable downstream cloning and sequencing of DNA and RNA containing unnatural building blocks. Dendrimeric structures that perform in sandwich assays to increase the number of fluorescent tags bound to an array by an individual target analyte. To validate these technologies for on-array use, the following specific aims will be met: 1. We will use the ink jet DNA array synthesizer to prepare target arrays incorporating these technologies, establishing a cycle of synthesis-test-evaluate-redesign-resynthesis that will allow in-house benchmarking of the innovative chemistries with respect to their ability to improve sample preparation and array performance. 2. We will synthesize a range of compounds needed to support the new technologies. 3. We will benchmark the new technologies with respect to their ability to improve: 3.1 Rates of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.2 Sensitivity of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.3 Selectivity with respect to single nucleotide mismatches. 3.4 Uniformity of response of multiple array elements targeted against different regions of a single analyte. 3.5 Sensitivity of detection of low abundance analyte targets, increased using dendrimeric structures. 4. We will then adopt innovative technologies to append AEGIS tags to natural samples in various sample preparation architectures 5. Last, we will develop a system for on-array validation of positive "hits" using sequencing using reversible terminators. PUBLIC HEALTH RELEVANCE This project, targeted for the National Human Genome Research Institute, is premised on the fact that innovative new chemistries are needed to improve the performance of array-based analysis of nucleic acids to meet the demanding specifications of biomedical researchers who do genomic science, demands that have increased as the performance of microarrays has improved. The Benner laboratory has invented approximately a dozen new chemical technologies that can be applied to each step of the genomic analysis, from sample preparation to array analysis. This proposal seeks funds to benchmark the improvements in array performance generated by these new technologies, improve them by a cycle of test and redesign, and provide them to the genomics community.